ONION SMUDGE 



J. c. WALKER UNIVERSITY OF WIcl.U!m..,h 
PH.D. THESIS 1 ^/^ 



Rtprinud from JOURNAL OF AGRICULTURAL RESEARCH 

Vol. XX, No. 9 : : : : Washington, D. C, February 1, 1921 




PCBUSHED BY AUTHORITY OF THE SECRET.\RY OF AGRICULTURE. WITH 
THE COOPERATION OF THE ASSOCIATION OF lAND-GRANT COLLEGES 



WASHINGTON : GOVERNMENT PRINTING OFFICE : 1921 



LlBflARY OF CONGRESS 

APR 6 1922 

DOCUivlcM;> ^.,.. .-.lOlv 



^^'fvJ^ 



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ONION SMUDGE 
By J. C. Walker 
Assistant Professor of Plant Pathology, University of Wisconsin, and Pathologist, Office 
of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant In- 
dustry, United States Department of Agriculture ' 

INTRODUCTION 

Smudge is a common disease of onions occurring both in the field and 
in storage or transit. It is confined for the most part to the bulbs and 
is characterized by dark green to black spots of variable size and shape 
on the outer scales. The spots may be homogeneous in appearance or 
may consist of numerous individual stromata scattered miscellaneously 
or arranged in concentric rings. The disease is most common on the 
white varieties of onions and damages materially the appearance and 
market value of the crop. The causal fungus has heretofore generally 
been known as Vermicularia circinans Berkeley, but as explained later 
in this paper it should more properly be termed Colletotrichum circinans 
(Berk.) Voglino. 

The present investigations have been carried on with special reference 
to the disease as it occurs in the districts of southeastern Wisconsin and 
northeastern Illinois where onion sets are grown. The growing of white 
onion "bottom sets" is an industry of considerable importance in these 
sections, and the methods used in growing and handling the set crop are 
often conducive to the excessive development of smudge during and 
immediately following har\'est. In this study attention has been given 
primarily to the mycological and physiological aspects of the causal 
organism, the relation of the parasite to the host tissue, the life history 
of the fungus with relation to the production of disease, and the develop- 
ment of remedial measures. 

THE DISEASE 

COMMON NAMES 

A number of common names have been used in American and Eu- 
ropean literature for this disease — namely, "onion Vermicularia" (j)^, 
" Vermiculariose " (29), "black spot" (7, 30), "scab" (//, 21), "an- 
thracnose" (7, 36, 37, j,S),and "smudge" {26). The name " anthracnose " 

1 This study was begun in the Department of Plant Pathology' at the University of Wisconsin in 1914. 
and the major portion was completed in 1917. Since the writer entered the Office of Cotton. Truck, and 
Forage Crop Disease Investigations in the latter year, observations have been extended to sections outside 
of Wisconsin. Grateful acknowledgments are expressed to Dr. L. R. Jones, imder whose inmiediate direc- 
tion the work has been done, and to Drs. J.J. Davis and E. M. Gilbert, who have given valuable aid and 
suggestions on the mycological phases of the problem. 

* Reference is made by niunber (italic) to "Literature cited.'' p. 719-721. 

Journal of Agricultural Research, Vol. XX, No. 9 

Washington, D. C. Feb. i, 1921 

wr KeyNo. G-218 

(68s) 



686 Journal of Agricultural Research voi.xx. No. 9 

has been much used up to the present time. However, since the symp- 
toms have httle in common with those of the more common anthrac- 
noses, and since it is believed that as simple and as descriptive a 
name as possible should be chosen, the name "onion smudge" is used in 
this paper to designate the disease, and this name is recommended 
for general usage. 

HOST PLANTS 

White varieties of the onion (Allium cepa) are the chief ones affected 
by smudge, but all varieties thoroughly tested have been found sus- 
ceptible to at least a slight degree. The disease also occurs on shallots 
{A. ascalonicum) and on leek (,4. porrum). It has never been found on 
garlic {A. sativum). 

HISTORY AND GEOGRAPHICAL DISTRIBUTION 

Onion smudge was first described in 1851 by Berkeley (4) in England, 
where it was found on the outer scales of a white variety. Subsequent 
reports of its occurrence in Europe have been made by Massee {17) in 
England, Bubdk (8) in Bo! emia, and Voglino ( jj) and Allescher (r ) in Italy. 

The first collection of this disease in America, made by Michener, was 
reported by Berkeley (5) in 1874. Since that time it has been re- 
corded in literature as occurring in Rhode Island {3), Connecticut 
{10, ig, 3j), New York (20, 22), New Jersey (ij, 25), Ohio (26), In- 
diana {21, 34), Illinois (jo), Wisconsin {23), and Alabama (2). Addi- 
tional data furnished by the Plant Disease Survey show that it has 
been present also in Massachusetts, Pennsylvania, Delaware, Mary- 
land, Virginia, Georgia, Louisiana, Texas, Minnesota, and Iowa. 

It is thus a disease of widespread occurrence; and, indeed, when one 
considers the fact that thousands of bushels of infected "bottom "sets 
are being shipped annually to all parts of the country and abroad, it is 
reasonable to suppose that its distribution is even more general than 
this summary indicates. 

DESCRIPTION OF SMUDGE (PL. 8o, 8l) 

The disease is confined entirely to the scales and the lower portions of 
the unthickened leaves which constitute the neck of the bulb. It first 
becomes manifest upon the appearance of minute stromata which form 
just beneath the cuticle of the host. These are dark green at first, be- 
coming black with age. Depending on conditions of infection, the indi- 
vidual stromata may be scattered miscellaneously over the surface of 
the bulb, or, as is more commonly the case, they may be congregated in 
smudgy spots around a few centers of infection. These spots are usually 
roughly circular and variable in size. They often coalesce and occa- 
sionally contain stromata arranged in concentric rings. Under moist 
conditions the stromata bear acervuli which contain prominent setae 
readily distinguished with a lens of low magnification. Cream-colored 
spore masses frequently form on these fruiting bodies. 



Onion Smudge 68 7 



Penetration of underlying dry scales by the fungus causes similar spots, 
which are commonly surrounded by yellowish borders. On the fleshy 
scales the disease first appears as minute, sunken, yellowish spots which 
gradually enlarge and often coalesce. As the disease progresses, the 
black stroma of the fungus usually appears; and, with the collapse of the 
host cells, spots very similar to those on the dry outer scales result. 
When the dark-colored stroma does not develop before the scale has 
entirely dried down, the affected portions appear as slightly raised, yel- 
lowish spots, giving to white onion sets an unnatural color which is 
almost as detrimental to their market value as the black, smudgy spots. 

The disease makes its appearance early in July under Wisconsin con- 
ditions, the fungus living on the outer dead scales and increasing in 
amount up to harvest time, when the outer two or three scales may be 
affected. From this time on it penetrates farther into the bulbs, progress 
depending upon en^Tronmental conditions. Badly diseased bulbs tend 
to sprout prematurely in storage. In most severe cases the fungus pene- 
trates the entire bulb and causes a complete collapse of the fleshy scales. 

The foregoing description applies to the disease as it appears on white 
onions. On colored varieties (red, yellow, and brown) the fungus is con- 
fined, with rare exceptions, to the neck of the bulbs where there is 
little or no pigment in the tissue, and the symptoms in these cases resem- 
ble closely those on the corresponding parts of the white varieties. 

On shallots the disease appears as smudgy spots very similar to those 
on onion and is confined to the outer leaves or scales. On leeks similar 
symptoms prevail. 

OTHER DISEASES LIKELY TO BE CONFUSED WITH SMUDGE 

Onion bulbs as they mature are subject to attack by a number of 
fungi which develop saprophytically on the dead outer scales and pro- 
duce symptoms which may easily be confused with those of smudge. 
The most common of these are two species of Macrosporium {Macro- 
sporium porri Ell. and M. parasiticum Thiim.) {33), and a species of 
Phoma, probably Phonia alliicola Sacc. and Roum. (24). The Macro- 
sporiums produce irregular, dark green spots which are due to ram- 
ification of the mycelium through the dead scales, but which lack the 
stromata and more or less regular outline of the smudge spot. In a 
moist atmosphere the fungi fruit and develop a dark green mold due to 
the production of conidia (PI. 81, F, G). In rare instances black peri- 
thecia of M. parasiticum are found on the outer bulb scales. Phoma 
produces small black pycnidia which are often difficult to distinguish 
macroscopically from the stromata of the smudge fungus. It is com- 
monly associated with M. porri (PI. 81, H). These two fungi commonly 
attack both white and colored varieties, and in the latter case the pig- 
ment in the outer scales is usually destroyed, giving a symptom which 
is known in the trade as "onion blotch." 



688 Journal of Agricultural Research voi. xx. No. 9 

Onion smut is sometimes confused with smudge, especially when the 
former occurs on mature bulbs. In such instances, however, smut usu- 
ally causes slightly raised, linear lesions which on colored varieties are 
commonly accompanied by more or less destruction of pigment. The 
exposure of the powdery spore mass upon breaking of the lesion estab- 
lishes the identity of the smut fungus. 

ECONOMIC IMPORTANCE 

The importance of smudge as a detriment to the onion crop may 
properly be considered from three standpoints — (i) that of reduction of 
market value as a result of marred appearance, (2) that of actual shrink- 
age of the bulbs in storage, due to fungus invasion, and (3) that of 
increased sprouting of onion sets during storage. Thaxter (jj) calls 
attention to the reduction of market value caused by smudge, citing 
an estimate by one grower of an actual loss of several thousand dol- 
lars to his crop in one season on this account. There is little doubt 
that marked spotting by this disease hampers greatly the disposal of white 
onions, since they are usually grown at a greater expense than colored 
varieties for a fancy trade which is prone to discriminate against dis- 
figured stock. Under prolonged storage smudge causes a distinct shrink- 
age of the bulbs and promotes premature sprouting. These last two 
factors are not usually of material importance on large bulbs, but they 
are of much significance with respect to onion sets. The latter are usu- 
ally harvested in August and September and kept in storage until March. 
The small bulbs are thus subjected to fungus invasion for several 
months, and data presented later in this paper show that in badly dis- 
eased sets the shrinkage may be doubled by smudge during this period. 

Sets which sprout badly during storage are a total loss to the owner, 
since they will not stand shipping and must be discarded. Much of the 
sprouting of white sets in storage is due to severe attacks by smudge. 
Experimental data in support of this statement are given later in this 
paper. 

It will be seen, therefore, that smudge is of greater importance than 
would be suspected from casual observation. In the Chicago district 
alone, where approximately i ,000,000 bushels of sets are grown annually, 
the aggregate loss due to shrinkage in weight and sprouting probably 
runs into many thousands of dollars. 

CAUSAL ORGANISM 
MORPHOLOGY 

The morphology of the causal organism has previously been discussed 
by Berkeley {4), Thaxter {33), Stoneraan (52), Stevens and True (jo), 
and Kempton (16). 

Mycelium. — The mycelium ranges from 2 to 8 microns in width, is 
septate and branching, varying widely with age as to color and size. It 



Onion Smudge 



689 



is at first hyaline \vith few septa, but later the walls thicken and take on a 
dark green color, oil droplets become more numerous, and septation is 
more frequent. 

Stromata. — By close intertwining of the thick-walled mycelial threads, 
dark green to black stromata, usually only a fraction of a millimeter in 
diameter and few to several hundred microns thick, are formed beneath 
the cuticle of the host (fig. i). On nutrient media these stromata 
commonly coalesce, forming a black stromateoid layer at the surface of 
the substrate. This coalescence sometimes occurs on the host, but 
more often the stromata remain distinct and are connected with one 
another by threads of the dark-colored mycelium. During protracted 
storage, or under poorly ventilated conditions, excessive stromata! 
development may occur (Plate 83, B). Thaxter (jj) describes large, 
somewhat flattened sclerotia, "jet black externally and white within," 




Fig. t. — Conidia and appressoria of Co/Ze/o/nVAum circinans. The fusoid conidia (C, D) germinate by one 
crmore germtubes. often becoming septate duringthe process(/7). Dark-colored, thick-walled appres- 
soria develop at the tip of the germ tubes, usually as the latter come in contact with the host cuticle (C. 
D). Subsequent germination of appressoria conunonly occurs (.4. C). Terminal or intercalary appres- 
soria-Iike cells, or chlamydospores, commonly develop within infected scales (B, £). Camera-lucida 
sketch. X 750. 

associated with the disease, though he does not definitely state 
that they are connected with the causal organism. The writer has 
never found bodies of this sort connected with the disease. On the other 
hand, sclerotia of Botrytis spp., which cause decay of onion bulbs and 
are commonly associated with smudge, compare favorably with his 
description. 

Appressoria or chlamydospores. — (Fig. i). These bodies are vari- 
able in size, dark brown in color, thick-walled, egg-shaped or roughly 
circular, usually temiinal but occasionally intercalary. In germination 
drops on glass slides they form most abundantly where the germ tube 
comes in contact with the slide and less commonly in the upper region 
of the drop. Under such conditions they measure 6.5 to 8 microns by 4 
to 5.5 microns. In Petri-dish cultures on various types of nutrient 
agar they are almost invariably produced at the tips of hyphae which come 
into contact with the glass surface. When "infection drops" containing 



690 



Journal of Agricultural Research 



viable conidia are placed on the surface of onion bulbs, appressoria or 
chlamvdospores are formed in contact with the scale. Later they send 
out germ tubes which penetrate the host. They are also commonly found 
within the tissue of affected scales. 

AcERVULi. — The fruiting bodies are fonned on the stromata which 
develop beneath the cuticle of the host. Short, hyaline conidiophores 
form in a palisade laj-er and rupture the cuticle of the host (fig. 2). One 
to several acervuli form on a single stroma. In the study of the morphol- 
ogy of the fruiting body the writer has found no evidence of a closed or 
partially closed receptacle, as described originally by Berkeley (4). Its 
true nature is more nearly in accord with the work of Stoneman {32), 
who found not a pycnidium but an open fruiting body. 




Fig. 2. — Acervulus of CoUetolrkkum circinans on artiScially inoculated onion scale. Note the develop- 
ment of the stroma in the subcuticular wall and the rupture of the cuticle by the formation of the 
palisade layer of the sporiferous hyphae. Camera-lucida outline. X 265. 

Setae. — Scattered throughout the acervulus are numerous setae 
arising from the basal stroma. They are thick-walled, dark -colored, 
o to 3 septate, upwardly attenuate, and 80 to 315 microns in length. 

Conidia. — -The conidia are borne acrogenously, being budded off one 
at a time. They are fusiform, continuous, hyaline to slightly ochraceous, 
somewhat curved, and obtuse at the very apex. Typically one prominent 
yacuole is present in the center of the conidium, but under some conditions 
the cytoplasm may contain many large vacuoles. As the spores are 
budded off from the conidiophores they form a cream-colored, somewhat 
mucilaginous mass on the top of the fruiting body. The spores vary 
from 14 to 30 microns in length and from 3 to 6 microns in width. A large 
majority, however, fall within the limits of 18 to 28 microns by 3 to 4 
microns. They germinate usually by one, but occasionally by two or 



Onion Smudge 691 



three germ tubes, whicTi are pushed out at any point on the surface. 
Septation of the spore commonly occurs during germination. 

PERIThecia^; — Stevens and True (jo) report the development of an 
ascigerous form on onion sets heavily infected with Colletotrichum cir- 
cinans and have referred the same to the new genus Cleistothecopsis. 
The writer has never been able to prove C. circinans to be connected 
with any ascigerous form found on onion. Stevens and True claim the 
connection between the perithecia of Cleistothecopsis and C. {Volutella) 
circinans on the following evidence: 

(i) they occurred on sets badly infected with the Volutella; (2) no other fungi or 
other types of mycelium were seen to be coimected with them; (3) when studied in 
various stages of development, the typical Volutella mycelium, which offers definite 
characters for recognition, was seen in organic connection with them, as illustrated 
in figure 18 (i), (4) the outgrowths from the perithecia are like those of the Volutella. 

This evidence is hardly sufficient to prove that the two forms are stages 
of the same fungus, especially since a large number of saprophytic or semi- 
saprophytic forms very commonly occur on the dead outer scales of 
onion bulbs and the differentiation of these from C. circinans on the basis 
of the characters of the mycelium is sometimes very difficult. The writer 
has, therefore, considered it advisable to use the binomial of the imper- 
fect form until cultures from a single ascus or ascospore of the ascigerous 
form are shown to be identical with C. circinans both as to morphological 
characters and pathogenicity upon onion bulbs. 

T.'^XONOMY 

The taxonomic questions involved in this study concern first, the 
proper position of the fungus in the present system of classification, and 
second, the possible identity of the organism with other described species. 

Berkeley (4) in the original description of the fungus refers to the 
fruiting body as a perithecium and places it in the genus Vermicularia, 
giving it the name Vermicularia circinans. Thaxter's (jj) description 
implies that the fungus has an open fruiting body, but he states that 
in the early stages of its development a "sort of membrane" extends 
over the basidia. Miss Stoneman (j2) describes a thick basal stroma 
bearing an open fruiting body. She also suggests that the characters 
of the fungus resemble more closely those of the genera Colletotrichum 
and Volutella than of Vermicularia. Voglino {35), believing the 
fruiting body to be an acer\'ulus, which would thus place the organism 
in the order Melanconiales, transferred the species to the genus Colletotri- 
chum. However, he gives no report of any study of the formation of 
the fruiting body. 

Stevens and True (jo) in discussing the fungus describe a sporodochium 
consisting — 

of a pseudoparenchymatous inner tissue covered by a continuous surf ace layer. . The 
young sporodochium eventually ruptures its covering membrane. . . In all cases the 
conidiophores are borne upon a raised superficial base which constitutes the sporodo- 



692 Journal of Agricultural Research voi.xx, N0.9 

chium, in contradistinction to the innate form of the acervulus which has no such 
base. The tubercular swelling, due to the massing of mycelium below and in the 
epidermis, partakes of sporodochial character also, and while this subepidermal part 
may not be regarded as constituting a true sporodochiura it serves to emphasize the 
tendency of the fimgus to produce such structures. . . The structure is a tubercle with 
a differentiated cortical outer layer. This outer layer ruptures and the tubercle 
develops as a sporodochium . . These facts exicude the fungus from Vermicularia and 
place it in the Tuberculariaceae under Volutella. 

In the discussion later in this paper on the relation of the parasite to 
the host it is shown that the development of the fungus commonly begins 
in the outer wall of the epidermal layer of host cells. As the cellulose 
becomes softened the hyphae multiply and a definite stroma forms with- 
in this softened cell wall. Mycelium penetrates the epidermal and 
underl3'ing cells, and if humid conditions prevail the stroma will soon 
occupy several layers of subepidermal cells. In good storage this process 
is comparatively slow, but during a protracted period, especially if the 
humidity rises considerably from time to time, the stroma commonly 
does acquire a thickness of several hundred microns. An examination of 
many sections has shown that regardless of the extent of its development 
the stroma is always covered by the cuticle of the host. At the instant of 
sporulation a palisade layer of hyaline hyphae interspersed with dark- 
colored setae arises from the stroma, and in this process the cuticle is rup- 
tured. This is shown to occur on stromata of widely different ages in 
figure I and Plate 83, B. It is to be noted in the first illustration that the 
stroma is of recent development, that it is confined to the outer wall of 
the epidermal layer, and that the cuticle has been ruptured only by the 
formation of the acer\'ulus. In the second illustration, although the 
stroma is much greater in extent, the host cuticle is still to be found in- 
tact except where it has been ruptured by the two acervuli. 

As pointed out by Saccardo {24, v. j, p. 221-222, 2jj), certain species of 
Vermicularia are characterized by imperfect or cup-shaped pycnidia, and 
such forms approach the genus Colletotrichum. Obviously it is often dif- 
ficult to determine the exact nature of the fruiting bodies, and as a result 
many forms belonging in Colletotrichum have been placed in Vermicu- 
laria. In the form under consideration there is no suggestion of pycnidial 
development at any time during the development of the fruiting body. 
On the other hand, it does fall \\ithin the limits of the genus Colletotri- 
chum. It is true that the basal stroma is much more highly developed 
than in many of the better-known species of this genus. However, well- 
developed stromata have been described in several species of this genus, 
including Colletotrichum anlirrhini by Stewart (jj) and C. cereale by 
Selby and Manns (27). In both cases the stroma develops beneath 
the cuticle, which is ruptured only upon the formation of the acervulus. 

It is qiute possible that a critical study of the closely related species 
classified at present in Vermicularia and Colletotrichum will lead to the 
separation into another genus of those forms which develop acervuli above 



Onion Smudge 693 



thick basal stromata. This question, however, is not within the province 
of the present paper. Those species of the Hyphales which are placed in 
the family Tuberculariaceae are characterized by the grouping together 
of the sporiferous hyphae in a superficial, conglutinate, sessile, or stipitate 
mass, known as a sporodochium {24, v. 4, p. 6j§, 682). As already pointed 
out, Stevens and True (jo) considered the fruiting body of the onion 
smudge organism to be of this nature and on that basis have transferred 
it to Volutella. In their description and figures, however, they seem to 
have interpreted the host cuticle as part of the so-called tubercle and 
thus as being of fungus origin. Were this true, the stroma would be super- 
ficial, and the fungus would properly belong to the genus Volutella. 
However, since the stroma is always subcuticular and the sporiferous 
hyphae are subcuticular in origin, the form is more characteristic of CoUe- 
totrichum than of Volutella. Here again it is obxdous that these two 
genera need more critical study before their limits can be satisfactorily 
defined. Meanwhile in the light of evidence just given, the writer con- 
siders it more suitable to use the name Colletotrichmn circinans (Berk.) 
Voglino for the onion smudge organism. 

The comparison of CoUetotrichiim circinans with other related species 
has been very limited in this investigation. The list of species of this 
genus which coincide closely with the one in question as to spore meas- 
urements and general characters is large and extends over a wide host 
range. Obviously the comparison of herbarium specimens is insufficient 
basis for final conclusions under the circumstances. Critical comparison 
has been confined to C. fnictus (S. and H.) Sacc, described as causing a 
fruit rot of apple. This species was originally described as a species of 
Volutella {28), but it was later transferred to Colletotrichum by Saccardo 
{24, V. 13, p. 1201)— 
on account of the black setae and the acervulus being originally subcuticular. 

Cross sections of apple fruits aff'ected with C. fructus and with C. circinans 
are compared in Plate 83, C, D. In both cases the development of the 
stroma beneath the cuticle, which is ruptured only upon the formation 
of the acervuli, is clearly shown. The former species was chosen for com- 
parative study because the spore measurements and general characters 
as previously described were closely similar to those of the onion smudge 
organism and authentic cultures were available. 

Cultures of the apple organism or diseased fruits were secured from 
Prof. C. R. Orton, State College, Pa., Dr. L. R. Hesler, Ithaca, N. Y., Dr. 
Charles Brooks, Washington, D. C, and Mr. G. A. Meckstroth, Columbus, 
Ohio. Cross inoculation on apple and onion showed that ColleMrichmn 
circinans was able to produce a rot of apple fruit similar to that produced 
by C. fructus (see Pi. 84, C). The formation of stromata and acer\'uli by 
both species on apple is shown in Plate 83, C, D. The rate at which the 
rot progressed, however, was uniformly slower in C. circinans. On onion, 



694 Journal of Agricultural Research voi.xx. N0.9 

C. jriiciiis developed on the dead outer scale of the bulb, but no evidence 
of further invasion as occurs with C. circinans was observed. Thus, the 
two species are distinct as to pathogenicit)-. 

Measurement of many hundreds of spores of several strains of both 
species produced on several substrates including the natural ones — 
namely, apple and onion — showed that the variations due to differences 
between strains and substrates along with differences due possibly to 
slight changes in environmental conditions precluded any distinction on 
this basis. The slight difference in the shape of spores shown in figure 3 
was quite uniform. The spores of Colletotrichuni fructus have walls 
nearly parallel throughout the middle half, and one end narrows much 
more abruptly than the other. 

A comparison of growth on potato agar gave further evidence as to 
the distinction of the two species. The chief points of difference in 
development on this medium are as follows: (i) Colletolrichum jriictus 





Fig. 3.— Spores of Colhlvlnihum /nidus (A) and C. circinans (B). Note the slifht difference in shape. 
In longitudinal section the walls of C. fructus are the more nearly parallel throughout the middle 
half, while at one end they converge more abruptly. Camera-lucida sketch. X 750. 

grows the more rapidly, (2) appressoria at the tips of hyphae coming in 
contact with the glass surface in plate cultures are absent in C. fructus, 
(3) the method of branching is quite distinct — that of C. circinans is 
dichotomous while that of C. fructus tends to be monopodia! in that 
nearly straight threads of mycelium, which become dark-colored very 
early and are greater in diameter, run out radially from the center of 
the colony and send out hyaline side branches of less diameter. Stromata 
develop at various points from these radial hyphae. This mode of 
growth gives a somewhat stellate macroscopic appearance to the colony, 
which differs from that of C. circinans, where distinctly radial hyphae 
are absent and stromata are scattered. This macroscopic difference is 
shown in Plate 84. 

Thus, although the morphological characters are only slightly variant, 
the two forms are considered distinct (i) because of difference in patho- 
genicity, {2) because of difference in spore shape, and (3) because of 
difference in type of colony on potato agar. 



Onion Smudge 695 



PHYSIOLOGY 
ISOLATION OF THE FUNGUS 

Pure cultures of the causal organism are readily obtained by the 
ordinary spore-dilution method. On potato-dextrose agar colonies 
appear in three to five days. Single spore strains were isolated from 
such cultures by means of the method described by Keitt (13)- Isola- 
tions thus made from many lots of diseased material collected in Wis- 
consin, Illinois, Ohio, Connecticut, and Louisiana have yielded strains 
which are closely similar in their behavior. 

CULTURAL CHARACTERS 

On potato agar (2 per cent dextrose) plates. — (See PI. 84,0, E.) 
The conidium germinates within 6 to 8 hours, sending out one to three 
hyaline germ tubes, which within 24 hours are many times the length 
of the spore. Colonies become macroscopic in about 2 days. The 
mycelium becomes somewhat thicker and denser in the center of the 
colony, while the younger hyphae around the outer edge are thin-walled 
and hyaline. Those branches of mycelium which come in contact with 
glass plates usually produce dark-colored, thick- walled chlamydospores 
or appressoria. Within 2 or 3 days stromata begin to form by abundant 
branching from a definite point in the mycelium, which finally results 
in a thick mass of hyphae. These hyphae assume an olivaceous color, 
and by the fourth day the dark green stromata are macroscopic in size. 
They form first at the center and later throughout the colony except at 
the extreme outer edge. Occasionally they are arranged in such a 
manner as to give the appearance of "fairy rings," but this is not a 
constant characteristic. The appressoria and the stromata give the 
young colony an olivaceous appearance. It becomes darker and almost 
black with age as the stromata become denser and more numerous and 
finally form an almost homogeneous stromateoid la3'er at the surface 
of the substrate. 

Bv the second day the colony shows a small amount of white aerial 
mycelium. This increases somewhat with age and later takes on a 
smoky gray appearance, masking the stromateoid layer to a certain 
extent. In from three to five days fruiting bodies are formed on the 
stromata at the center of the colony, and they continue to develop as 
the colony grows. Conidia are produced in abundance in most strains, 
accumulating in cream-colored or pinkish masses on the fruiting bodies. 

The colony will continue to grow to an indefinite size if space and 
nutrients are available. A diameter of about 25 mm. is reached in 
seven days at room temperatures. 

On potato agar (2 per cent dextrose) slants. — Growth is similar 
in most respects to that on plates. Aerial mycelium tends to be more 
abundant. Mycelium does not, as a rule, extend deeply into the agar to 
form stromata. As the culture dries out the aerial mycelium forms a 



696 Journal of Agricultural Research voi. xx, No. 9 

dense mat over the surface of the culture, its color usually becoming 
slightl}' brownish with age. Spore masses often appear above this layer 
of mycelium. 

On other media. — The growth of the fungus was studied on 25 
kinds of artificial media, including beef broth agar, corn meal agar, oat 
agar, apple agar, synthetic agars, vegetable agars, cooked vegetables, and 
fresh vegetable tissues. The character of growth on the various media 
used was so uniform and so closely parallel to that on potato agar that a 
separate description for each is unnecessary. The most noticeable dif- 
ference was that correlated with the supply of sugar in the medium. 
Where dextrose was omitted in the formula growth and sporulation were 
very scanty, and the stromata were few in number and widely scattered. 
On onion and apple agars made up without dextrose this difference was 
less marked, probably on account of the presence of a considerable 
amount of sugar in the plant tissues used. On synthetic agars ' with 
sugar added in the form of maltose, dextrose, lactose, and sucrose 
copious growth took place with no evidence of preference for any one of the 
carbohydrates used. Cooked bean pod, onion scale, carrot, potato, and 
rice supported good development of the organism. On fresh onion and 
apple, however, the growth was much retarded, and on fresh potato 
and carrot it was very scanty. Stevens and True (jo) report retarded 
growth on onion broth agar made with red or yellow varieties. The 
writer has found equally vigorous development on agar made from red, 
yellow, and white types of onion. 

RELATION OF TEMPER.\TURE TO GROWTH 

Potato agar plates inoculated with mycelium or conidia of the fungus 
were kept at temperatures ranging from 1° to 35° C. The rate of growth 
was determined by measuring the diameter of the resulting colonies or 
thalli from day to day. In order to increase the accuracy of the results 
Petri dishes of equal diameter containing equal amounts of agar were 
used. In order to overcome the influence of variations in relative 
humidity prevailing in different incubators the later experiments were 
modified by placing the Petri dishes in moist chambers first and then 
exposing them to the desired temperature. It was found after many 
trials that the best comparative data could be secured at four to six 
days. The growth was slight at 1°, almost negligible at 2°, but an 
appreciable amount occurred at 8° to 10° during a period of 10 to 14 
days. Above this point the rate of growth increased rapidly, reaching 
the optimum at about 26°. At 31° to 32° little or no growth occurred 
on potato agar. The growth at various temperatures on this medium 
at the end of 6 days is represented graphically in figure 4. 

^ Formula for sviithetic agar used: Sugar, loo gm.; peptone, 20 gm.; anunonium nitrate, 10 gm.; mag- 
nesium sulphate, 2.5 gm.; potassium nitrate, 5 gm.; acid potassium phosphate, 2.5 gm.; calcium chlorid, 
0.1 gm.; agar, so gm.; neutralized with normal sodium hydroxid. 



Onion Smudge 



697 



A similar study of growth in tubes of onion decoction was made, with 
essentially parallel results. The optimum on this medium appeared to 
be slightly higher (27° to 29° C.) and slight growth occurred at 31°. 

SPORE GERMINATION 

Relation of medium. — For the studies upon spore germination a 
few drops of the liquid medium to be used were placed in Van Tieghem 
cells. A suspension of conidia in the same liquid was made, and a drop 
of this was transferred to cover glasses, which were then inverted over 
the cells and partially sealed with 
vaseline. The preparations were 
placed in Petri dishes and exposed 
to the desired conditions. For 
some purposes open drops on glass 
slides placed in Petri dishes lined 
with moistened filter paper were 
more suitable. 

A comparative study of spore 
germination in distilled water, 
onion decoction,' onion leaf ex- 
tract,^ onion scale extract,' soil extract 
and soil decoction ^ was made. 

At room temperature germination in favorable liquid medium began 
within 5 to 6 hours. At 24 hours practically all viable spores had germi- 
nated. The percentage of germination in the drops was determined by 
averaging the counts of several microscopic fields. The results of these 
tests are summarized in Table I. 

Table I. — Effect of various media upon spore germination of Colletotrichum circinans 




Fig. 4. — Relation of temperature to growth of 
CoUetotrickum circinans on agar plates. 



(sterilized and unsterilized),'' 



Medium. 


Percent- 
age of ger- 
mination. 




60 

95 
95 
10 

99 




















Onion scale extract, diluted with distilled water i to 10 



500 cc. distilled water steamed one hour, filtered, and sterilized. 
; (green) crushed and the sap extracted by squeezing through 



' Onion decoction: loo gm. onion scale i 

2 Onion leaf extract: Fresh onion leavi 
cheesecloth. 

' Onion scale extract: Fresh onion scale crushed and the sap extracted as in onion leaf extract. 

• Soil extract: 500 gm. black loam soil was supported in a glass funnel by excelsior and absorbent cotton. 
500 cc. of tap water were poured over the soil; the filtrate was collected twice, and each time it was poured 
over the soil. The third filtrate was divided into two parts; one part was left unsterilized and the other 
part was sterilized in tubes at 15 pounds pressure for M hour. 

*Soil decoction: 500 gm. of black loam soil, to which had been added 500 cc. of distilled water, was steamed 
at 15 pounds pressure for 3 a hoiu". The liquid was filtered through filter paper and sterilized in tubes at 
iC pounds pressiu-e for J^a hour. 



698 



Journal of Agricultural Research 



Vol. XX, No. 9 



The striking outcome of this comparison is the marked retardation in 
unsteriHzed soil extract and the complete inhibition in onion leaf and 
onion scale extract. Even when the last two were diluted with 10 parts 
of water no germination occurred. As pointed out in a previous note 
by the writer {38), further experiments have sho^\^^ the presence of at 
least two distinct substances in onion tissue which are probabl)- respon- 
sible for inhibition of spore germination. A more detailed study of this 
phase and its relation to the parasitism of the fungus will be included in 
another paper. Cooked soil extract, soil decoction, and onion decoction 
stimulate germination and promote rapid growth of the germ tubes. 
It is evident that the cooking of the onion scale removes or destroys the 
substances which are unfavorable for spore germination. 

Relation of temperature. — Since conidia were found to germinate 
well in distilled water, this medium was used for studies of the effect of 
temperature on spore germination. A large number of tests were run at 
a gradation of temperatures ranging from 1° to 35° C. Spores were 

found to germinate between the 
limits of 4° and 32°. Appressoria 
developed in germination drops 
throughout the same range of tem- 
perature. At 35° to 37° slight swell- 
ing of the spores took place, giving 
them the appearance of "involution 
forms," but normal germination 
did not occur. Figure 5 is a 
graphic representation of the effect 
of temperature as indicated by per- 
centage of conidia germinating in 
Best germination occurred at about 20°, but 





















r 


J 


\ 








J 


/ 






\ 






/ 








1 


\ 


/ 


/ 










\ 




5 K 




? a 


J s 


S- c* 


P <35 



-Relation of temperature to spore gennina- 



tion of CoUeloiricku 



distilled water at 12 hours 

good germination occurred between 13° and 25°. 

The temperature range for spore germination thus coincides closely 
with that of fungous growth. The point of optimum development is 
comparatively high, and this fact is significant in exolaining the occur- 
rence of the disease in the field. 



EFFECT OP DESICCATION 

In order to interpret more fully the development of the disease in the 
field and the overwintering of the causal organism, the effect of desicca- 
tion on conidia and stromata was studied in tlie laboratory. 

On conidia. — Studies were made on conidia as they occur (i) in 
masses on the fruiting body on the host, where they are embedded in 
the mucilaginous material which surrounds them, (2) in similar masses 
on potato agar, and (3) in water suspension, where the spores are sepa- 
rated from one another, approximating to some extent conditions as 



Onion Stmidge 699 



they occur in nature when spores are disseminated by meteoric water. 
Diseased onions bearing spore masses were brought in and allowed to 
dry out gradually in the laboratory, and the viability of the spores was 
tested from time to time. Ordinarily a large percentage lost their 
vitality within 2 weeks, but in some cases good germination occurred 
after 7 weeks. A small percentage of conidia from spore masses pro- 
duced on potato agar and exposed to similar conditions germinated after 
4 months. Spores in water suspension allowed to dry out on glass 
slides were very sensitive to desiccation, little or no germination occurring 
after 24 hours. It is evident, then, that the conidia are sensitive to 
desiccation except when they remain in waxy masses on the host, in 
which condition a small percentage will remain viable through extended 
unfavorable periods. These results aie in accord with the findings of 
Hasselbring {14) for the somewhat closely related fungus Gloeosporium 
fructigenum, causing the bitter-rot of apple. 

On stromaTA. — The stromata of the fungus are capable of withstand- 
ing very long periods of desiccation. Test tube cultures of the fungus 
on a large number of media were kept at room temperature for a period 
of two years. Since the tubes were not plugged very tightly with cotton 
the cultures dried out completely within four or five months. The 
vitality of the fungus in this desiccated condition was tested by adding 
sterile melted potato agar to the tube and slanting them until the fresh 
medium hardened. Vigorous growth characteristic of the fungus re- 
sulted from the cultures originally made on potato, beef broth, carrot, 
com meal, oatmeal, and onion agars, steamed rice and bean pods, and 
fresh potato and onion plugs. The fungus was no longer viable on 
synthetic agar, steamed potato, carrot, onion, and fresh carrot. Since 
spores lose their vitality in such a long period of drying, it may be in- 
ferred that the fungus lived through this extended period of desiccation 
by means of the stromata which developed in the substrate. It is to be 
expected from these results that the stromata which develop in the scales 
of the host are capable of carrying the fungus over long periods of un- 
favorable climatic conditions. 

EFFECT OF FREEZING 

On conidia. — Spores in water suspension exposed to freezing tem- 
peratures are killed within a few hours. Fresh spore masses also are 
very sensitive to low temperatures, but if they are allowed to dry out 
before being exposed to freezing temperatures they will withstand such 
temperatures for a month or more. In order to test the resistance of 
conidia to the freezing weather of the entire winter period, infected 
onion bulbs bearing spore masses were placed out of doors in a weather 
instrument shelter at Madison, Wis., on December 7, 1915. Germina- 
tion tests showed a high percentage of these conidia to be viable at this 



yoo Journal of Agricultural Research voi.xx. N0.9 

time. Tests made on January 22, 191 6, showed that by this date all 
the spores had been killed. A similar experiment was carried out at 
Madison in the winter of 1919-20. Infected bulbs bearing abundance 
of spore masses were placed out of doors in October, 191 9, and protected 
from rain and snow. A few viable spores were obtained on March 20, 
1920. Thus, a few conidia may \\-ithstand Wisconsin winters if suffi- 
ciently protected, but probably few, if any, live over under field condi- 
tions. 

On sTROMata. — Agar cultures containing abundant stromateoid de- 
velopment were kept out of doors during the winter months at Madison, 
Wis., during which period there was much severely cold weather. In 
all cases the cultures were found to be viable at the end of this time. 
Stromata on onion scales have also been exposed in this region during 
the winter period, and in every case they withstood the severe freezing 
temperatures. 

It is to be expected from the foregoing data that spore masses with- 
stand short intervals of dry weather during the summer and furnish 
ready inoculum upon the return of moist conditions. During extended 
periods of unfavorable conditions, however, the stromata serve best to 
perpetuate the fungus. 

PATHOGENICITY 

Inoculation experiments were performed on plants at various stages 
of growth from young seedlings to mature bulbs. 

Sterilized greenhouse loam soil was inoculated by spraying with a 
water suspension of spores at the time of sowing onion seed. Three 
hundred seeds of White Globe variety were planted in the inoculated 
soil and the same number in uninoculated soil. Ten days later, as the 
cotyledons were coming through the soil, the attack of the fungus became 
evident by the rapid collapse of the succulent tissue at any point on the 
young shoot. Acervuli of the fungus were present and continued to 
develop on the diseased portions of the plants. Fifteen days after 
sowing, 64 out of 123 plants in the inoculated pot were diseased, whereas 
all of the 161 plants in the control pot were healthy. This experiment 
was repeated several times, and in each case where sterilized soil was 
inoculated a high percentage of the seedlings were killed. When un- 
sterilized greenhouse soil was used the injury was greatly reduced, 
the competition of other soil organisms evidently greatly limiting the 
activity of the smudge fungus. Moreover, damping off of this sort due 
to smudge has never been noted in old onion set fields, other factors, 
such as low temperature at this early part of the season, probably limit- 
ing the activity of the fungus. 

Leaves of half -grown plants were sprayed with a spore suspension and 
kept in a moist chamber for 24 to 48 hours. The fungus developed and 
fruited on the lower leaves, which had reached a stage of "physiological 
old age," but this never occurred on vigorously growing leaves. 



Onion Smudge 



701 



The disease was produced many times by means of artificial inoculation 
of healthy mature onion bulbs with suspensions of spores from pure 
cultures, and the fungus was readily reisolated. A summary of these 
inoculations is given in Table II. In certain cases when bulbs kept in a 
closed chamber were thus inoculated, the experiment was unsuccessful. 
It was found in such instances that although the spores were capable of 
germination in water, they did not germinate in the drops on the bulbs. 
The inhibitive effect of the volatile oil of onion on spore germination 
was mentioned earlier by the writer (j8). An accumulation cf this 
substance when several onion bulbs are placed in the small space in a 
moist chamber may possibly account for this lack of germination. 
Further studies on this point will be described in a later paper. 

More nearly uniform results were secured when sterilized soil was inocu- 
lated bv spraying with a spore suspension and healthy bulbs then inserted 
in this medium for a week or 10 days. The outer scales usually became 
uniformly infected in 7 or 8 days (see PI. 81, C). WTien the bulbs were 
removed and placed in storage, typical invasion of the underlying scales 
occurred. 

Table II. — Summary of inoculation and greenhouse experiments on onion bulbs 





lation 
No. 


Date of in- 
oculation. 


Method of 
inoculation. 


Inoculated. 


Controls. 


Type of inoculatiou. 


Num- 
ber of 

used. 


Percent- 
age in- 
fected. 


Num- 
ber of 
days 
before 
first 
note of 
disease. 


Num- 
ber of 
onions 
used. 


Per- 

age in- 
fected 


In moist chambers . 


I, a 

I 
27 

29 

3- 
35 
44 
49 
59 
61 

3 

23 

4S 

I 50 


July 24 

7 

Jan. 8 

20 

20 

July 20 

Nov. 21 

Dec. 16 

Apr. 12 

26 

Aug. 26 

Dec. 3 

Nov. 30 

Dec. 16 


Spray 

. ..do 

...do 

...do 

...do 

...do 

...do 

...do 

...do 

...do 


5 
I 

5 
5 
5 
4 
5 
5 
3 
2 

10 
5 

15 



888.g°88cg°88888 


12 

6 

5 
13 

"; 

6 
18 

'"'g' 
8 




I 

5 
5 
5 

I 

5 
5 



10 
5 

15 
9 











































In general, then, the fungus assumes the r61e of a weak parasite. 
Actively growing portions of the plant are not attacked except in young 
seedlings grown under certain conditions. In the field the fungus is 
confined to the outer leaves or scales, the cells of which are dead or essen- 
tially functionless. As the plant approaches maturity the dry outer 
scales of the bulb are invaded, but the nonnal fleshy scales are not 
affected at this time. A few cases have been noted where the fungus 



702 



Journal of Agricultural Research 



attacked growing scales which were being parasitized by the smut 
fungus, Urocystis cepulae, but apparently a weakening of the plant 
is necessary before actual invasion of the growing parts occurs. Fol- 
lowing harvest there is a gradual invasion of the dormant cells of the 
fleshy scales of the bulb as previously described. The progress here 
is usually slow, but in a moist, warm environment there may be a more 
rapid invasion, resulting in decay of the resting central bud of the onion 
set. 

RELATION OF THE CAUSAL ORGANISM TO THE HOST TISSUE 



Onion bulbs from which the thin outer scales had been removed 
were placed in moist chambers. Inoculum consisting of a suspension 
of spores from pure culture in sterile distilled water was applied to the 
uninjured surface of the exposed scales, either in drops by means of a 
platinum loop or as a spray from an atomizer. 

For the study of penetration a razor section was cut tangentially 
from the surface of the scale directly beneath the infection drop so as 




Fig. b.—Colletotrichum circiiums: Stage of penetration of epidermal cell of onion scale at 66 hours alter 
inoculation. Camera-lucida sketch. Approximately X 430. 

to contain the epidermis with a few layers of the immediately underlying 
cells. This was examined directly in Mo in a water mount, the absence 
of chlorophyll in the host cells making clearing and staining unneces- 
sary. For the study of the relation of the fungus to the host tissue 
following penetration, pieces of inoculated scale as well as of naturally 
infected fleshy scales were fixed in Fleming's medium fixative, washed, 
dehydrated, embedded in paraffin, and sectioned according to standard 
methods of procedure. In some material a satisfactory differentiation 
of fungus and host was secured by omitting the bleaching of the micro- 
tome sections (commonly done after using a fixative containing osmic 
acid), which left the mycelium black, and then counterstaining the 
host cell walls with orange G. In other cases the iron haematoxylin 
and Delafield's haematoxylin stains gave satisfactory results. 

PENETRATION 

Under optimum conditions germination occurs within lo hours and 
appressoria are formed, either sessile or at the end of short germ tubes. 
Usually the appressorium is flattened to some extent on the side adja- 



Onion Smudge 



703 



cent to the cuticle. The penetration tube is formed from the flattened 
side of the appressorium and penetrates the cuticle directly (iig. 6, 7). 
Blackman and Welsford (6) have pointed out that solution of the host 
cuticle by invading fungi has never been fully demonstrated; they 
explain the invasion of bean leaf cuticle by Botrytis cinerea as mechani- 
cal in nature. The mode of penetration in onion smudge was not 
definitely ascertained, but it seems highly probable that the germ tube 
from the adhering appressorium might pierce the thin cuticle by means 
of mechanical pressure. 




SUBSEQUENT DEVELOPMENT 

The fungus hyphae, after penetration, develop first between the sub- 
epidermal wall and the cuticle, which is rather elastic in nature and 
can be raised considerably without being 
ruptured. Figure 6 illustrates the extent of 
invading germ tubes at 66 hours after inocu- 
lation. The nature of the penetration tube 
and the subsequent development beneath the 
cuticle are shown in figure 7. In certain 
other anthracnose fungi — namely, CoUdotri- 
chum lagenarium as reported by Gardner {iz), 
C. lindemuthianum by Dey {11), and Gloc- 
osporiinn jructigcnutn by Hasselbring {14) — 
the penetration tube has been described as 
invading the cell wall directly. This is also 
the case in Botrytis cinerea on bean (6), al- 
though the germ tube in this instance does 
sometimes grow horizontallv beneath the 
cuticle. The softening of the subcuticular 
wall in the case of onion smudge soon be- 
comes apparent by its swelling and taking on a laminate appearance. 
The hyphae grow through and between the laminae (fig. 8) and by rapid 
development soon form the beginning of the stroma previously described. 
The swelling of the outer wall eventually involves the entire lumen of 
the epidermal cell. Although the g-reatest amount of fungus growth at 
this stage takes place just beneath the cuticle, occasional hyphae pene- 
trate underlying cells. As the hyphae attack these cell walls, softening 
and lamination take place as in the subcuticular wall, while penetration 
is seemingly accomplished partly by means of chemical action and 
partly by mechanical pressure. The relation of mycelium to the 
parenchyma cells just beneath the epidermal layer is also shown in 
figure 8. In the case of bulbs inoculated in moist chambers the collapse 
of invaded cells was not rapid, and there was no evidence noted of 
injury to the cells in advance of the mycelium. 



Fig. 7. — Cross section of epidermis, 
showing early stage of penetration 
by Collelolridmm circmans. Note 
the empty appressoria with myce- 
lium still wedged between the cuti- 
cle and the subcuticular wall. 
Material fixed 72 hours after inocu- 
lation. Camera-lucida sketch. 



704 Journal of Agricultural Research Voi.xx. N0.9 

Under ordinary storage conditions, the progress of the fungus is 
closely parallel to that just described, except that the progress is much 
slower under this diflferent environment. As described before, the first 
macroscopic symptom of invasion from spots on the dry outer scale to 
the underlying fleshy scale is a small, yellowish, slightly sunken area. 
This usually increases in size very slowly in well-ventilated storage. A 
cross section of one of these, spots is illustrated in Plate 83, A, and a de- 
tailed drawing from a similar section is shown in figure 9. The fungus 
develops extensively at first just beneath the cuticle, and the softening 
and lamination of the subcuticular wall is very slight. As invasion pro- 
gresses, hyphae penetrate this wall directly, evidently by chemical solu- 
tion rather than mechanical pressure, since the ca\'ity is slightly larger 
than the mycelium and there is no sign of bulging of the wall before 
penetration is achieved. The collapse of cells beneath the epidermal 





Fig. 8.— Cross section of epidermis (.4 ) and underlying parenchi-ma cells (B) of onion scale inoculated with 
a suspension of Colletotrichum circinans spores and kept in a moist chamber at room temperature. 
Note softening and lamination of cell walls by the invading hyphae. Material fixed five days after 
inoculation. Camera-lucida sketch. .4, X 308; B, X 350. 

cell takes place before any appreciable invasion of hyphae occurs. In 
the section shown in Plate 83, A, two layers beneath the epidermal layer 
have collapsed, while only an occasional hypha is to be found beneath 
the subcuticular wall. There is no evidence of softening of the cell wall. 
Morever, in such lesions mycelium has never been found in the walls or 
lumina of turgid living cells. This suggests that ejther the cells are 
killed in advance of the hyphae or only slight invasion of the wall leads 
to their collapse. This slow invasion, which prevails even after the cells 
have become functionless, is surprising in view of what occurs when bulbs 
are inoculated in moist chambers. Is it possible that the volatile oil 
present in the onion scale is influential in checking the advance of the 
fungus ? 

Under moist conditions and optimum temperature the stroma develops 
very rapidly in the subcuticular wall, and acervuli are formed in five to 



Onion Smudge 705 



six days after inoculation. This condition is shown in figure 2. In other 
cases where sporulation is postponed through lack of proper environ- 
ment the stroma continues its growth more slowly and eventually in- 
volves a larger portion of the scale. The cuticle, however, remains 
intact on the exterior and normally is not ruptured until the palisade 
layer of conidiophores is formed. A cross section of a scale which had 
been held in poorly ventilated storage several months is shown in Plate 
83, B. Acervuli were produced upon exposure to proper conditions for 
sporulation. Note that the cuticle is still present outside the extensive 
stroma, except where it has been ruptured by the sporiferous hyphae. 

FACTORS IN THE PRODUCTION AND PROGRESS OF THE DISEASE 

OVERWINTERING OF THE CAUSAL ORGANISM 

The experiments already reported on the effect of desiccation and 
freezing upon conidia indicate only a remote possibihty that the fungus 
lives through the winter in this form under Wisconsin conditions. The 
stromata, on the other hand, are capable of withstanding protracted 
periods of drouth or freezing temperature. In order to confirm the 
supposition that the fun- 

gus actually over^vinters ^^s>,_^rX__^,^^^^^ 
and IS widely disseminated ^•^j^^iri^S^^y^^^^"'^^ 

in this latter fonn, four lots ^ ^^■•j 

of heavilv infected bulbs Fic,-<:ross section of oni^on scale naturally infected wU 

Coiletotrtchum fircjjiowj. showing the mycelium developing 
were placed out of doors at first just beneath the cuticle and later penetrating the sub- 

Madison, Wis., on Decem- fUticul-waU Camera-ludda sketch. X 450. (This phase 

' ' IS illustrated further m PI. 83. A). 

ber 7, 1915.. One lot was 

left in an instrument shelter near the surface of the ground, and the 
remaining lots were buried in the soil at depths of 2, 4, and 6 inches, 
respectively. Spore masses were present on this material at the begin- 
ning of the experiment, and germination tests showed a high percentage 
of the conidia to be viable at this time. 

On January 22, 1916, examination of spores from the bulbs placed 
in the instrument shelter showed that they had completely lost liability 
by that date. The four lots of bulbs were examined on April 12, 1916. 
Those which had been buried in soil readily produced conidia in abun- 
dance upon exposure to humid conditions at room temperature. The 
material kept in the instrument shelter had dried out considerably 
during the winter and, though much slower to respond, eventually 
proved to be viable by the production of spores. A similar experiment 
conducted during the winter of 1916-17 yielded confirmatory data. 

It is to be expected that infected scales from the crop of the pre\ious 
season furnish a source of abundant inoculum for initial infection of 
the growing crop. This, combined with the fact that in most onion- 
growing sections it is the common practice to grow this crop successively 



7o6 Journal of Agricultural Research Voi. xx. No. 9 

on the same field for many years, results in a heavy infection of a large 
part of the white set crop annually. Examination of a large number of 
fields in Wisconsin and IlHnois has revealed the fact that "clean" white 
sets are secured as a rule only from land growdng its first crop of onions. 
In a majority of cases the second crop of white sets is badly infected. 

In all fields examined where the first crop of onions was being grown, 
an occasional bulb infected \nth smudge was found. A satisfactory 
explanation of these original infections has never been reached. Many 
possible means of introduction of the fungus from neighboring infected 
fields immediately suggest themselves, such as manure, farm imple- 
ments, man and farm animals, drainage water, and wind, and undoubtedlv 
some of these often do play a part in the distribution of the disease. 
The possibility of seed as a carrier is also to be considered in this con- 
nection. Although smudge has never been found attacking the floral 
parts of the plant, it is conceivable that those seed umbels which fall over 
and come in contact with the soil before harvest might become infected 
or be the means of introducing bits of infected scales to the seed. It 
should be noted in this regard that the spores of onion smut, a disease 
which is also confined to the bulb and leaves of the plant and in fact does 
not attack onion seed plants, have previously been found on onion seed 
samples (9, 18). 

One experiment was perfonned on the relation of seed to the dis- 
semination of the fungus. Samples of six varieties of seed were sown 
in pots of sterilized soil in the greenhouse on December 5, 191 6. On 
January 16, 1917, all the seedlings were examined. Fruiting bodies of 
Colletotrichum circinans were found on the outer scales of two seedlings 
of the White Globe variety and of one seedling of the Queen variety. 
No other signs of the disease were found. The identity of the fungus 
was confirmed by isolation of pure cultures and comparison with authentic 
strains. Two subsequent plantings of the same sample of White Globe 
seed were made, but no further sign of the disease was found. The small 
amount of the fungus occurring in this experiment is not surprising, 
since only a very limited amount of infectious material can be expected 
to be seed-borne. However, although the evidence at hand indicates 
that the fungus is carried on seed to some extent, further data are 
necessary before a final conclusion on this point can be made. 

REL.'iTION OF TEMPER.ATURE TO INFECTION AND TO DEVEI.OPMENT OF 
THE DISEASE 

Studies of the relation of temperature to the germination of conidia 
and to their subsequent growth have shown the optimum to be about 
20° C. for the former and 26° for the latter. The range in each case, 
however, is wide. Accordingly a set of experiments was started for the 
purpose of determining the range and optimum temperature for infection. 

Sterilized loam soil in glass or glazed crock jars was inoculated with 
a water suspension of spores. Healthy white onion sets were then 



Onion Smudge 707 



inserted in the soil; and the jars, each covered with a glass plate, were 
placed in incubators running at temperatures ranging from 5° to 32°. 

In the first experiment 10 onions were placed in each of four jars 
which were placed in incubators held at 5°, 13° to 14°, 23°, and 28° to 31° 
C, respectively. The extent of the disease on the various lots at this 
time is shown in Plate 82. It was apparent that infection took place 
very slowly at 13° to 14°, while that at 28° to 31° was shghtly less ad- 
vanced than at 23°. 

In the second experiment jars containing 10 onions each were held at 
, 5° to 6°, 9° to 10°, 14° to 15°, 17° to 18°, 20° to 21.5°, 22° to 23°, 26° to 
27°, and 30° to 32° C. They were allowed to remain for 17 days before 
examination. At the end of this period, no infection had taken place 
at 5° to 6°, a very slight infection at 9° to 10°, and as the temperature 
rose the amount of disease increased up to 26° to 27°, at which point it 
was greater than in any of the other jars. At 31° to 32° it was slightly 
less than at 26° to 27°. A third experiment confirmed the results of the 
first two. 

Infection takes place and the disease progresses, then, at or above 10° 
C, but it is quite evident that for very rapid development a temperature 
of 20° or above is needed. Since the fungus develops in the soil prior to 
infection, the range of soil temperature during the growing season is 
undoubtedly an important factor in determining the severity of the 
disease. 

PRODUCTION AND DISSEMIN.\TION OF CONIDIA 

After the appearance of the first stromata on the bulbs, subsequent 
spread of the disease is eff'ected to a considerable extent by conidia 

Sporulation does not take place except under fairly humid conditions- 
In order to determine the range of temperature at which fructification 
may occur, infected scales were placed in Petri dishes lined with moist- 
ened filter paper and exposed in incubators running at a range of temper- 
atures from 2° to 28° C. Abundant sporulation occurred within 36 hours 
at 20° to 28°. The process was much retarded at lower temperatures, 
though a few spores were formed at 2° to 3° after several days. 

Under optimum conditions for spore production the conidia accumu- 
late on top of the acerv^uli, forming gelatinous masses which remain in- 
tact among the setae. Exposure of portions of scales bearing fresh spore 
masses over sterile agar plates has yielded no indication of spore dis- 
charge. The mucilaginous material surrounding the spores appears to 
dissolve partly when a spore mass is placed in water, and the conidia 
thus become separated. 

It is thus to be expected from the nature of the fungus that warm, 
rainy weather is especially favorable for the development of smudge, 
since high humidity promotes the production of spores, and meteoric 
water, especially in the form of spattering rain drops, is important for 
their dispersion and dissemination. 



7o8 



Journal of Agricultural Research 



Vol. XX. No. 9 



CORRELATION OF CLIMATIC CONDITIONS WITH THE DEVELOPMENT OF TH3 
DISEASE IX 1 91 5-1 6 

Plots of white onion sets were grown in 191 5 and 191 6 on land which 
had previously produced many successive crops of onions and where 
the smudge organism was known to be present in the soil. Soil tem- 
perature records were taken at a depth of i to 2 inches during part of 
the 1915 season and most of the 1916 growing season. The daily mean 
soil temperatures and rainfall for these seasons are represented in figure 



Q35 






^ S 



i 










. . U,.,. ._...^, .T^/aiy^S'T ^^k .;, ....-_....... 




~:y^z:f% w ^' - -i-^ ■ - ^-- 


-M%r4-----ii t i 


p ^f -J- - ^ 

1 ._..... 


1 




^ ^ _ ^ _ -J L L- J- ^ _^ - 


. -- ^pi.liL-^ -.- =4 -^-!— 4ii^ -.-» hIii -, — T^-Hi -5-mins lifeiri EkC 












1 __ _ .— 


nVJi^ 4. frJirlfflh- .- i - 4^_LJIta 



Fig. 10.— Chart from data collected at Racine. Wis., during 191S and 1916, showing the daily mean soil 
temperature at a depth of i to 2 inches, and the rainfall. The horizontal broken line represents the 
optimtmi temperature for infection and development of the disease as indicated by controlled 
experiments, the broken vertical line the date of first observation of the disease in 191s. and the heavy 
vertical line the first appearance of the disease in 1916. 

ID. The rainfall records included here are compiled from data taken 
at the Racine (Wis.) post office, approximately 3 miles from the onion 
set plots. The progress of the disease between the time of its first sea- 
sonal appearance and harvest is described for these two seasons, since 
they represent distinctly different conditions which had varying effects 
upon the progress of the disease. 

IN" I915 

On June 28 a very few dark green stromata were found, but no acer- 
vuli or setae had developed. The soil temperature mean was now well 



Onion Smudge 709 



above 20° C. and remained between 20° and 27° for most of the time 
until harvest. On July 2 a few scattered acervuli were found. A 
slight precipitation was recorded on July 2, 2 inches on July 4, 0.02 inch 
on July 5, and 1. 1 7 inches on July 7. Following this rainy period there 
was a marked increase in number of acervuli noted on July 10. A slow 
rain fell during most of July 14 and part of July 15. On July 15 the 
disease was prevalent above the bulbs on the unthickened portions of tne 
outer leaves which comprise the "neck." These infections were clearly 
the result of spores spattered upon these portions from the bulb scales 
by rain a few days previously. The rainy weather, which prevailed until 
har\'est, about August 10, resulted in continued spread and development 
of the disease, so that the white sets were all badly spotted by the latter 
date. Further observations showed that the development of the disease 
in other fields followed closely that noted in the experimental plot. The 
infection in practically all cases, however, was confined to one or two of 
the outer dry scales, the fungus being unable to attack the fleshy scales 
previous to harvest. On the yellow and red varieties the fungus was 
very abundant on the uncolored portions of the leaves at the neck, but 
the highly colored bulb scales remained entirely free from it. This has 
been the usual observation with the colored types. 

IN 1916 

The month of July, 191 6, was extremely warm and dry as contrasted 
with cool, moist weather of the same period inigis. The soil tempera- 
ture mean passed 26° C. on July 2 and remained above that point for the 
rest of the month. In fact, for a large portion of that period it was well 
above 32°, the maximum temperature for growth of the fungus on potato 
agar. No signs of smudge were found until July 8. The extent of the 
disease at this time was very meager, only a few acervuli being noted. 
It is probable that the dry weather preceding this date checked the 
fungus, in spite of the fact that the soil temperature was favorable. 
Aside from 0.03 inch precipitation on July 8, 0.45 inch on July 20, and 
0.14 inch on July 31 , no rain fell during the rest of the month. More- 
over, the soil temperature was well above the maximum for development 
of the disease. On July 13 but very little smudge could be found. 
On July 22 no further development was noted. The moisture from 
the shower of July 20 disappeared very rapidly from the upper 2 
inches of soil because of the extreme heat. A rainy period occurred 
on August 3, 4, and 5, and following this Macrosporium porn 
and Phoma alliicola developed rapidly. Smudge increased but very 
slowly, however, probably because of the scarcity of viable spores. 
Another heavy rain fell on August 9 and 10, and the weather then 
remained clear until after har^'est on August 23. At the latter date 
the bulbs were examined carefully, and in general the sets were only 
moderately infected. The disease was confined for the most part to the 
portions of the bulbs below the surface of the soil, while the abundant 



yio Journal of Agricultural Research voi. xx, N09 

infections on the necks which were so conspicuous in 191 5 were almost 
entirely absent. 

To summarize, the disease progressed most rapidly during the last 
part of the growing season of 1915, with the mean temperature range 
between 20° and 30° C, accompanied by sufficient rainfall to promote 
abundant spore production and dissemination as well as subsequent 
infection. On the other hand, development was materially checked in 
1 91 6 by extreme heat, together with lack of precipitation during July. 

RELATION OF ENVIRONMENT DURING CURING TO THE DISEASE 

The onion set crop is usually harvested in early August. The tops 
are twisted or clipped and the small bulbs are placed in shallow crates 2 
or 3 inches deep. These are stacked in the field in piles with temporary 
roofs, where they are allowed to cure foi several weeks. Usually the 
fungus is well established upon the outer scales of the bulbs before they 
are pulled, and thus further invasion is dependent largely upon the 
en\dronmental conditions which prevail during the curing and storage 
periods. 

The respiratory functions of the living cells m the bulbs continue after 
the sets are pulled, and there is, in consequence, some accumulation of 
moisture. This is counteracted in part by the use of shallow crates 
which are exposed to natural air currents. In bright, windy weather 
the bulbs cure rapidly, while rainy or humid weather retards the process 
and favors the progress of the disease. A number of experiments were 
conducted during 1916, 1917, and 1918 to determine the effect of varied 
amounts of external moisture during the curing period upon the develop- 
ment of the disease. 

Experiment 1. — On August 15, 191 6. a crate of white sets was taken 
from the general run of the crop which had been harvested on August 9 
at Racine, Wis. The outer scales were badly spotted with smudge, and 
in some cases the second scale had been invaded. After removal to the 
laboratory the bulbs were sprinkled with water while in the crates. 
After two days a portion of this lot (5^ pounds) was dried for 24 hours at 
45° to 52° C. and the remainder (14K pounds) was given no further 
treatment. Both lots were placed under cover in a shallow crate, where 
they were exposed to good conditions for further natural curing. They 
were later placed in a well-ventilated onion warehouse held at about 
35° to 40° F. On January 13, 191 7, both lots were examined. Most 
of the outer dead scales present at harvest time had sloughed off during 
storage, and in the dried sets the fungus had advanced very little from 
these original infections. In the naturally cured sets, however, the 
fungus, probably aided by the greater excess of moisture present, had 
invaded several underlying scales, and these sets were badly spotted 
even after the outer scales were removed. The sets in each lot were 
then sorted into three classes — (i) free from disease, (2) slightly diseased, 



Onion Smudge 



711 



(3) badly diseased. The result of this classification is given in Table III, 
and samples from the dried and the undried lots are shown in Plate 85, 
A, B. 



Table 111.— Relation 


of artificial cxcring 


to the develop 


ment of onion smudge 






Condition at end of storage period. 


Treatment. 


Percentage 
free from 
disease. 


Percentage 
slightly 
diseased. 


Percentage 

badly 
diseased. 




7 
56 


29 
36 


64 




8 







Experiment 2. — On August 30, 1917, several bushels of white onion 
sets were secured from a field where the crop had been har^^ested on 
August 16 and placed in stacks of shallow crates. The weather had 
been clear during this intervening period, and good natural conditions 
for curing had prevailed. Smudge was prevalent on the outer scales of 
the sets at this time. In order to test the effect of exposure to moist 
weather on the progress of the disease, a portion of this lot in the crates 
was sprinkled with water daily for one week, approximating roughly what 
often occurs when a rainy period comes during harvest. After one 
week a part of the moistened lot was placed in a kiln drier, where the 
temperature was held at 100° to 120° F., until the bulbs were thor- 
oughly dried. The remainder of this lot was allowed to dry naturally 
under cover. All the sets were then stored in a standard onion storage 
house. Samples taken from a moistened and an unmoistened crate on 
October 10 are shown in Plate 85, C, D. Marked increase in the amount 
of smudge was very noticeable within a few days after moistening was 
begun. On January 14, 191 7, the amount of smudge was estimated by 
classifying several hundred bulbs from each of the three lots into either 
of two classes, namely, (i) those free from smudge or only slightly 
diseased and (2) those so badly diseased as to impair their market 
quality. The results are given in Table IV. 

Table IV. — Effect of varied conditions at harvest on the amount of smudge on storcdonion 

sets 

Condition at end of 
storage period. 



Best natural curing 

Exposed to moist conditions after harvest 

Artificially dried after exposure to moist conditions 




712 



Journal of Agricultural Research 



This experiment shows (i) that even under what may be considered 
very good weather conditions for natural curing a considerable amount 
of smudge will develop; (2) that exposure to moist weather for a week 
after harvest practically doubled the amount of smudge; and (3) that 
thorough artificial drying immediately after such exposure counteracts 
the effect of excessive moisture. 

Experiment 3.-^The sets used in this experiment were from a late 
sowing and consequently were not har\-ested until September 14, 1918. 
Smudge was prevalent on the extreme outer scales of a large percentage 
of the bulbs at this time. Five bushels were placed in shallow crates 
in the kiln drier, in which the temperature was maintained at 100° to 
120° F. One crate was removed at the end of one day, a second at the 
end of two days, and the remaining three on the fifth day. Three 
untreated crates used in the experiment were allowed to cure in a cov- 
ered pile in the field with the remainder of the crop. On September 30 
they were removed to a standard onion warehouse, where they were 
stored during the winter with the artificially dried lots. On March 5, 
1 91 9, when final notes were taken, a comparison of the artificially cured 
and field-cured lots was secured by estimating the percentage showing 
any signs of smudge after sets had been milled to remove the loose 
scales.' The results are given in Table V. 

Table V. — Amount of smudge on artificially cured and field-cured onion sets at the end 
of the storage period 



Nature of treatment. 



Artificially dried. 

do 

do 

do 

do 

Field-cured 

do 

do 



Average of artificially dried crates. 
Average of field-cured crates 



Length of 
treat- 
ment. 



showing 

any 
signs of 
smudge. 



The foregoing experiments clearly establish the importance of moisture 
as a factor in the advance of the disease during the curing and storage 
periods. They also indicate that artificial curing immediately following 
harvest greatly checks the progress of the disease as compared with 
natural field-curing. 

' sets through a fanning mill as they are taken from storage 



Onion Smudge 713 



RELATION OF STORAGE CONDITIONS TO THE DISEASE 

The study of the disease in storage has been directed toward the 
solution of three problems: (i) The importance of smudge as a cause 
of premature sprouting of sets; (2) the extent of shrinkage, if any, 
which can be brought about during the storage of onion sets; and (3) 
the amount of new infection or actual spread from diseased to healthy 
bulbs occurring during the holding period. While the data on these 
points are by no means complete and the factors involved in the progress 
of the disease during the storage period by no means fully studied, the 
experiments here reported upon throw some light on the matter. 

Observations on the first two questions were made in a standard onion 
set warehouse at Morton Grove, 111. In practice, onion sets are stored 
in crates about 4 inches deep with slatted bottoms, piled so as to allow 
a I- to 2-inch space between each two crates to facilitate circulation of 
air. Sets are placed in storage during September and October. The 
temperature is gradually lowered, following seasonal changes, until it 
approaches 0° C. (32° F.), an attempt then being made to hold it slightly 
above this point. During extremely cold weather some artificial heat 
in the house is necessary to prevent freezing, while ventilation is con- 
stantly needed to remove excessive moisture. 

The experiments were carried on during the winter of 1918-19. The 
extremely mild weather during this season prevented the temperature 
of the house from being held as close to 0° C. as is commonly the case, 
while, on the other hand, ample opportunity for ventilation was afforded. 
Continuous records of temperature and relative humidity were secured 
by means of a Friez hygro-thermograph. The temperature gradually 
lowered during October and November, the minimum temperature reach- 
ing 0.5° C. (33° F.), on November 23, while the maximum temperature 
commonly reached 12.7° C. (55° F.) during this period. During Decem- 
ber, January, and February the temperature fluctuated between 0.5° 
and 7.2° C. (33° and 45° F.). The relative humidity varied between 65 
per cent and 85 per cent during October and November, while through- 
out the remainder of the period it seldom went above 75 per cent and 
not often below 60 per cent. 

RELATION OF SMUDGE TO SPROUTING 

Two lots of onions were used in these experiments, and, since they 
differed somewhat as to time of maturity and method of handling, they 
are here considered separately. 

Experiment i. — Bulbs averaging about i inch in diameter were 
selected from a lot of white sets harvested earl}' in August and brought 
into storage on August 22, 1918. Two groups were secured, one con- 
sisting of 49 bulbs badly spotted with smudge and the other containing 
47 perfectly healthy sets. The two lots had thus been grown and han- 
dled alike and presumably differed only as to infection with smudge. 



714 



Journal of Agricultural Research 



Vol. XX. No. 9 



They were carried through storage and examined on February i8, 1919. 
The results are given in Table VI. 



Table VI. — Relation of smudge to sprouting of onion sets 
EXPERIMENT I 



storage 



Condition of bulbs. 


Total 
number of 
bulbs used. 


Number 
sprouted. 


Percentage 
sprouted. 


Healthy 


47 
49 


14 
26 


29.7 

5o-° 







Experiment 2. — The sets used in this experiment were sown late in 
the spring and consequently were not har\'ested until about September 
14, 1918. They were allowed to cure in the field in the normal manner 
until September 30, when they were placed in storage. Three average 
crates were selected at this time and kept under observ^ation. At har- 
vest time smudge was prevalent only on the dry outer scales of the sets, 
but during the storage period it gradually penetrated the underlying 
scales. When a final examination was made on March 5, 1919, it was 
clear that in nearly every case where the fungus had penetrated deeply 
the bulb had sprouted and had thus become worthless. A typical 
example of this condition is shown in Plate 81, D. An estimate of the 
amount of sprouting actually due to or intimately associated with smudge 
was secured by counting 100 to 200 bulbs in each crate. The results 
are given in Table VII. 

Table VII. — Relation of smudge to sprouting of onion sets in storage 
EXPERIMENT 2 



Crate No. 


1 smudge. 


Total 
percentage 
sprouted. 


Total 
percentage 
sprouted 
and showing 
advanced 
stage of 
smudge. 




165 
197 
1 48 


75 
75 
72 


6.0 
9.6 

2. 


6. 




9.6 
•7 








74 


5-S 


5-4 







It is not to be construed from these data that smudge is always the 
chief cause of premature sprouting of onion sets in storage, since un- 
questionably other factors may often be entirely responsible. One of 
these, the rteckrot decay of the scales, commonly produces a similar effect. 
It is apparent, however, that the invasion of the bulb scales by the smudge 
fungus brings about some physiological change which promotes growth 
of the previously dormant bud. 



Onion Smudge 



715 



Economically this factor has considerable value, since bulbs which 
sprout before the end of the storage period are usually a total loss. 

RELATION OF SMUDGE TO SHRINKAGE OP SETS IN STORAGE 

In order to secure bulbs as nearly comparable as possible except for 
presence or absence of smudge, healthy and diseased sets averaging 
about I inch in diameter were selected from a general lot of white sets 
which had been harvested in early August, properly field-cured, and 
placed in storage on August 22, 191 8. Four lots of 25 bulbs each were 
secured which showed heavy smudge infection but no signs of any other 
disease. Three lots of 25 each were selected which appeared to be per- 
fectly healthy. All lots were weighed on October 15. Two diseased lots 
and one healthy lot were kept in the warehouse throughout the experi- 
ment under conditions previously described. In order to secure a high 
relative humidity a special temporal y chamber was made in the ware- 
house and lined with moistened burlap. Thus, a relative humidity of 
90 to 95 per cent was maintained at a temperature close to that of the 
main warehouse. Two diseased and two healthy lots were placed in this 
chamber for approximately four weeks and then' removed to the main 
warehouse room. The several lots were weighed on December 30, 1918, 
and on February 18, 1919. The results of the experiment are given 
in Table VIII. A constant increase in shrinkage of diseased sets ever 
healthy sets was to be noted. Before the end of the experiment sprouting 
had occurred in most of the lots, and, as was to be expected, was more 
prevalent in diseased than in healthy lots. Sprouting and the complica- 
tion of contaminating parasites should be considered; but, since the 
former is seemingly enhanced by the disease and the latter is not serious 
in these cases, there is reason to believe that smudge is responsible in 
large measure for the increase in shrinkage. 

Table VIII. — Relation of smudge to shrinkage of onion sets in storage 



Condition of bulbs. 



weight, 

Oct. IS 

1918. 



Condition at end 



Ordinary storage. 



Healthy. 
Diseased . 



Exposure to high rela- 
tive htunidity for 4 
weeks, followed by c 
dinary storage. 



5 sprouting. 
8 sprouting; i in- 
fected with blue 
mold. 



Healthy I do. 

do ] do. 

Average shrinkage of dis- 

eased lots , 

Average shrinkage of 
healthy lots 



7i6 



Journal of Agricultural Research 



Vol. XX, No 9- 



SPREAD OF SMUDGE IN STORAGE 

It has been claimed that smudge spreads from infected to healthy 
bulbs in storage (77, 29). It is to be expected that under unusually 
moist conditions this might occur. However, since considerable mois- 
ture is necessary for sporulation and infection, the conditions which 
prevail in good storage houses are not conducive to rapid spread of 
the disease. Several experiments have been conducted during the course 
of this investigation in which healthy bulbs have been marked and mixed 
in lots of badly diseased sets. A summary of these experiments appears 

in Table IX. 

Table IX. — Spread of smudge in storage 



Experi- 
ment 

No. 


storage conditions. 


Length 
of ex- 
peri- 
ment. 


Num- 
ber of 
healthy 
bulbs 
used. 


Condition at end of experiment. 


I 


Standard onion warehouse 

do 


Days. 
154 
103 

66 
208 

36 


34 
40 
20 
20 
20 


All healthy. 

2 bulbs showed slight infection. 

All healthy. 

Do. 
6 showed slight infection. 


3 

4 
S 




do 

Moist chamber at room tem- 
perature . 



It was found that there was little or no spread of the disease under 
ordinary storage conditions or in a cool cellar. In a saturated atmos- 
phere some infection of healthy bulbs occurred. In practice, then, some 
spread from diseased to healthy bulbs is to be expected where sets are 
exposed to rain or very humid atmosphere such as might occur during 
the curing period. However, with fairly dry sets kept in cool, well- 
ventilated storage new infections are probably negligible. 

CONTROL OF THE DISEASE 

The control of this disease is obviously connected closely with the 
handling of the crop at or immediately following har\'est. 

In 1 91 5 a spraying experiment was conducted on a plot of white sets 
at Racine, Wis. The development of the disease in this plot has been 
described on pages 708-709. Various schedules were used with 4-4-50 
and 8-8-50 Bordeaux mixture plus soap, 4-50 copper sulphate, and i-io, 
1-16, and 1-32 lime sulphur. The sprays were applied upon the bulbs 
and necks of the plants. Contact with the soil probably reduced the 
disinfecting property of the chemicals, and their adhesiveness was limited 
by the nature of the scales and leaves of the onion. No beneficial results 
were secured even where the first application was made before the first 
signs of the disease appeared and where the spraying was continued at 
intervals of three to eight days until har^-est. The complete failure of 



Onion Smudge 717 



this experiment was sufficient to show that sprays could not be used 
successfully for the control of smudge. 

Dusting of the sets in the crates at harvest time with lime or sulphur 
has been suggested by Thaxter (33). In 191 6 and 191 8 dusting ex- 
periments with lime, sulphur dust, and dry Bordeaux powder were con- 
ducted without any positive results. This is to be expected, since, as 
a rule, the outer scales of the bulbs became infected before harvest 
and a disinfectant applied externally could hardly prevent further 
invasion of underlying scales. 

The importance of thorough curing and prevention of exposure to 
humid conditions after harvest has been emphasized by Thaxter {33), 
Clinton {10, p. jjj) , IMassee (17), and Stevens and True (jo). The experi- 
ments reported on the effect of drying of bulbs at hars'est have shown 
that rapid dehydration of the outer scales at this time checks further 
invasion by the fungus to a large degree. Observations in the field by 
the writer during the years 191 4 to 1920 indicate that even the best 
natural curing weather to be expected in the Middle West is not sufficient 
to do more than partially check the disease on seriously infected fields. 

Artificial curing offers a possible measure of control for smudge, and, 
as already pointed out (57) , preliminary experiments indicate that neckrot 
can also be checked by this treatment. Extensive control experiments 
carried on in the Chicago district in 191 8 have shown that thorough dry- 
ing very soon after harvest is necessary in order to check smudge materi- 
ally. In the set-growing district a large portion of the crop is gtown on 
contract to be delivered at a central warehouse as soon as it has cured 
sufficiently. The expense involved in this treatment would almost 
necessitate that they be dried at a central point, preferably at the place 
of storage. Therefore, in order to handle the large quantity received, a 
fairly rapid process of drying would be essential. 

Further experimental work is necessary before artificial drying can be 
recommended as a general practice, and the results of control experi- 
ments are reserved for later publication. In the meantime, the most 
applicable remedial measures consist in prompt harvest and the best 
use of natural climatic conditions in curing the white onion set crop, 
including all possible protection from moist weather. This should be 
followed by storage in a well-ventilated warehouse held as nearly as pos- 
sible at 33° to 36° F. 

SUMMARY 

(i) Smudge is one of the most common diseases of white onion sets in 
Wisconsin and Illinois. 

(2) It occurs also on shallot {Allium ascaloniciim) and leek {A . porriim) . 

(3) The disease was first described by Berkeley in England in 1851 and 
is now widely distributed in Europe and America. 



7i8 Journal of Agricultural Research voi.xx. No. 9 

(4) Smudge is confined to the scales and neck of the bulb, where 
it causes dark green to black spots. On fleshy scales it appears as sunken 
yellowish spots which enlarge slowly, coincident with gradual shrinkage 
of the scale. On colored varieties the disease is confined to unpig- 
mented portions of the outer scales of the neck of the bulb. 

(5) Spots on the outer scales of bulbs due to Macrosporium porri, 
M. parasiikum, Phoma alliicola, and Urocystis cepulac may be confused 
with smudge. 

(6) Smudge becomes detrimental to the onion crop as a cause of (i) 
the reduction of market value of white varieties, (2) shrinkage in storage, 
and (3) premature sprouting of sets in storage. 

(7) A detailed description of the morphology of the causal organism, 
Collctoirichum circinans (Berk.) Voglino, is given. The ascigerous 
form, Cleistothecopsis circinans, has been described by Stevens and True, 
but complete proof of its connection with Collelotrichum circinans is 
lacking. 

(8) Inasmuch as the causal organism produces a subcuticular 
stroma and a well-defined acervulus, the species is classified in the l\Ielan- 
coniales as Collelotrichum circinans (Berk.) Voglino. A comparative 
study of the latter with C. jrtictus (S. and H.) Sacc. was made. 

(9) The characteristic growth of the organism on culture media is 
described. 

(10) Growth on potato agar takes place between 2° and 32° C, while 
the optimum is about 26°. 

(11) Spore germination is stimulated in soil decoction, onion decoc- 
tion, and sterilized soil extract, as compared with that in distilled water, 
while it is reduced in unsterilized soil extract and entirely inhibited in 
onion leaf or scale extract. 

(12) Spore germination occurs within the range of 4° and 32° C, while 
the optimum temperature is from 20° to 26°. 

(13) Conidia are very sensitive to desiccation except when in spore 
masses, in which condition a small percentage retain vitality for four 
months or more. Stromata are very resistant to desiccation, retaining 
vitality for two years or more. 

(14) Conidia are sensitive to freezing temperatures, but dried spore 
masses may withstand this environment for a month or more. Stromata 
are capable of withstanding several months of freezing weather. 

(15) The fungus is pathogenic upon the scales of mature bulbs, but 
does not attack actively growing parts of the plant with the exception 
of young seedlings, upon which it may cause "damping off" under certain 
greenhouse conditions. 

(16) Spores germinate and appressoria form within 10 to 12 hours. 
The infection tube is pushed from the side of the appressorium adjacent 
to the host cuticle directly through the latter. The mycelium then de- 
velops for a time between the cuticle and the subcuticular wall, raising 



Onion Smudge 719 



the former and eventually causing a softening of the latter. In bulbs 
inoculated in moist chambers the fungus progresses fairly rapidly, caus- 
ing softening and lamination of the walls and the gradual collapse of the 
cell. The stroma involves the subcuticular wall at first and later the 
underlying cells, but the cuticle remains unbroken until the acervulus is 
formed. The process of invasion under storage conditions is essentially 
the same but much slower. 

(17) The fungus overwinters as stromata in infected scales. 

(18) Infection occurs at or above 10° C, but progress is very slow 
below 20°; the optimum is about 26°. 

(19) Conidia are produced abundantly under moist conditions and at 
temperatures between 20° and 30° C. They are disseminated chiefly by 
meteoric water, especially spattering rain. 

(20) The disease develops most rapidly in the field when the mean soil 
temperature range lies between 20° and 30° C. and is accompanied by 
abundant rainfall. Extremely hot, dry weather in July checks progress. 
Presence of moisture favors the progress of the disease during the curing 
period, whereas artificial drying of sets immediately following har^'est 
checks it. 

(21) Smudge tends to promote premature sprouting and increases 
shrinkage of sets in storage. The disease may spread from bulb to bulb 
in the crate under very moist conditions, but in proper storage this factor 
is neghgible. 

(22) The important measures of control are protection of the har- 
vested crop from rain, rapid and thorough curing, and provision of well- 
ventilated storage at about 33° to 36° F. 

LITERATURE CITED 
(i) Allescher, Andreas. 

1898-1901. FUNGI iMPERFECTi . . . 1016 p. Leipzig. (Rabenhorst, L. Kryp- 
togamen-Flora von Deutschland, Oesterreich und der Schweiz. Aufl. 
2, Bd. I, Abt. 6.) 

(2) Atkinson, G. F. 

1897. SOME FUNGI FROM ALABAMA . . . BuL Cornell Univ. (Sci.), v. 3, no. i, 
50 p. Bibliography, p. 2. 

(3) Bennett, J. L. 

1888. PLANTS OF RHODE ISLAND, BEING AN ENUMERATION OP PLANTS GROWING 
WITHOUT CULTIVATION IN THE STATE OF RHODE ISLAND. 128 p. 

Providence, R. I. 

(4) Berkeley, m. j. 

1851. [a NEW ONION DISEASE.] Jn Gard. Chron., 1851, no. 38, p. 595, 2 fig. 
(5) 

1874. NOTICES OF NORTH AMERICAN FUNGI. In Grevillea, V. 3, no. 25, p. 1-17. 
Continued article. 
(6) Blackman, V. H., and WelsFord, E. J. 

1916. STUDIES IN THE PHYSIOLOGY OF PAR.\SITISM. II. INFECTION BY BO- 

TRYTis ciNEREA. Ill Ann. Bot., V. 30, no. 119, p. 389-398, 2 fig., pi. 
10. Literature cited, p. 397. 



720 Journal of Agricultural Research voi. xx, N0.9 

(7) Britton, W. E., and Clinton, G. P. 

[igi8.] SPRAY CALENDAR. Conn. Agr. Exp. Sta. Bui. 199, p. 51-98, illus. 

(8) BubIk, Fr. 

1904. IN BOHMEN IM JAHRE 1902 AUFGETRETENE PFLANZENKRANKHEITEN. 

In Ztschr. Landw. Versuchsw. Oesterr., Jalirg. 7, Heft 10, p. 731-741. 

(9) Chapman, George H. 

1910. NOTES ON THE OCCURRENCE OF FUNGOlIS SPORES ON ONION SEED. Mass. 

Agr. Exp. Sta. 22d Ann. Rpt., 1909, p. 164-167. 

(10) Clinton, G. P. 

1904. DISEASES OF PLANTS CL-LTIV.\TED IN CONNECTICUT. Coim. AgT. Exp. 
.Sta. 27th Ann. Rpt., 1902/03, p. 279-370, pi. 9-28. 

(11) Dey, p. K. 

1919. studies in the physiology of par.\sitism. v. infection by col- 

LETOTRICHUM LINDEMUTHI.\NUM. In Ann. Bot., V. ^Il,, no. 131, p. 
305-312, pi. 21. References, p. 311. 

(12) Gardner, M. W. 

191S. ANTHRACNOSE OF CUCURBITS. U. S. Dept. AgT. Bul. 727, 68 p., 15 fig., 
8 pi. Literature cited, p. 65-68. 

(13) Halsted, BjTon D. 

189I. REPORT OF THE BOTANICAL DEPARTMENT. N. J. Agr. Exp. Sta. Ilth 

Arm. Rpt., 1890. p. 323-453, illus. 

(14) Hasselbring, Heinrich. 

1906. THE APPRESSORIA OF THE ANTHRACNOSES. hi Bot. Gaz., V. 42, nO. 2, 

p. 135-142, 7 fig- 

(15) Keitt, G. W. 

1915. simple technique for ISOLATING SINGLE-SPORE STRAINS OF CERTAI.»J 

TYPES OF FUNGI. In Phytopathology, v. 5, no. 5, p. 266-269, i fig- 
16) Kempton, F. E. 

19I9. ORIGIN AND DEVELOPMENT OF THE PYCNIDIUM. In Bi-t. Gaz., V. 68, nO. 
4. P- 233-261, pi. 17-22. 
(17) Massee, George. 

1903. A TEXT-BOOK OP PLANT DISEASES CAUSED BY CRYPTOGAMIC PARASITES. 

ed. 2, 472 p.. illus. London, New York. 
(iS) MuNN, M. T. 

1917. NECK-ROT DISEASE OF ONIONS. New York State Agr. Exp. Sta. Bul. 
437. P- 361-455. II pl- Bibliography, p. 45°-455- 

(19) Orton, W. a. 

1903. PLANT DISEASES IN THE UNITED STATES IN 1902. U. S. Dept. Agr. Year- 
book, igo2, p. 714-719. 

(20) 

1907. PLANT DISEASES IN 1906. U. S. Dept. Agr. Yearbook, 1906, p. 499-508. 

(21) OsNER, George A. 

1917. ADDITIONS TO THE LIST OF PLANT DISEASES OF ECONOMIC IMPORTANCE IN 

INDIANA. In Proc. Ind. Acad. Sci., 1916, p. 327-332. 

(22) Peck, Charles H. 

1881. REPORT OF THE BOTANIST. In 34th Ann. Rpt. N. Y. State Mus. Nat. 
Hist., p. 24-5S, 4 pi. 

(23) RussELL, H. L. 

1915. REPORT OP THE DIRECTOR. PLANT DISEASE SURVEY. Wis. AgT. Exp. 

Sta. Bul. 250 (Rpt. 1914), p. 33-39, fig. 14-17- 

(24) Saccardo, p. 

1884-1913. SYLLOGE FUNGORUM . . . v.3,1884; V. 4, 1886; V. 22, 1913. Patavii. 



Feb. I. isai OnioH Smudge 721 

(25) ScHWARZE, Carl A. 

:9i7. THE PARASITIC FUNGI OP NEW JERSEY. N. J. Agr. Exp. Sta. Bui. 313, 
226 p., 1056 fig. 

(26) Selby, a. D. 

1910. a brief handbook of the diseases of cultivated plants in ohio. 
Ohio Agr. Exp. Sta. Bui. 214, p. 307-456, 106 fig. List of plant diseases 
referred to in this publication, p. 1-7. 

(27) and Manns, T. F. 

1909. STUDIES IN DISEASES OF CEREALS AND GRASSES. Ohio Agr. Exp. Sta. 

Bui. 203, p. 187-236, illus., 14 pi. 

(28) Stevens, F. L., and Hall, J. G. 

1907. AN APPLE ROT DUE TO VOLUTELLA. In Jour. Mycol., V. 13, no. 89, p. 
94-99, 6 fig. 

(29) ■ 

1 9 10. DISEASES OF ECONOMIC PLANTS. X, 513 p. , illus. New York. 

(30) and True, Esther Y. 

1919. BLACK SPOT OF ONION SETS. 111. Agr. Exp. Sta. Bul. 220, p. 505-532, 

19 fig. 

(31) Stewart, F. C. 

1900. AN ANTHRACNOSE AND A STEM ROT OF THE CULTIVATED SNAPDRAGON. 

N. Y. State Agr. Exp. Sta. Bul. 179, p. 105-110, 3 pi. 

(32) StonEman, Bertha. 

1898. A COMPARATIVE STUDY OF THE DEVELOPMENT OP SOME ANTHRACNOSES. 

In Bot. Gaz., v. 26, no. 2, p. 69-120, pi. 7-18. Bibliography, p. 114-117. 

(33) Thaxter, R. 

1890. REPORT OP . . . MYCOLOGIST. In Conn. Agr. Exp. Sta. Ann. Rpt., 1889, 
p. 127-177, 3 pi. 

(34) Van Hook, J. M. 

1911. INDIANA fungi. In Proc. Ind. Acad. Sci., 1910, p. 205-212. 

(35) VOGLINO, p. 

1907. I FUNGHI PARASSITI DELLE PIANTE OSSERVATI NELLA PROVINCIA DI TORINO 

E REGiONi viciNE NEL 1906. In Ann. R. Accad. Agr. Torino, v. 49, 
p. 175-202. 

(36) Walker, J. C. 

1917. STUDIES UPON THE ANTHRACNOSE OF THE ONION. (Abstract.) In 

Phytopathology, v. 7, no. i, p. 59. 

(37) 

1918. CONTROL OF NECK ROT AND ANTHRACNOSE OF ONION SETS. (Abstract.) 

In Phytopathology, v. 8, no". 2, d. 70. 

(38) 

1918. NOTES ON THE RESISTANCE OF ONIONS TO ANTHRACNOSE. (Abstract.) 

In Phytopathology, v. 8, no. 2, p. 70-71. 



PLATE 80 

Onion smudge: 

Onion sets (White Portugal variety) naturally infected with Colleiotrichum cir- 
cinans. Collected on August 27, 1919, several weeks after harvest, at Morton Grove, 
111. Photographed September 23, 1919. Note in the three lower bulbs the small 
sunken spots in the fleshy scales which mark the early stages of invasion of the living 
tissue. Natural size. 

(722) 



Onion Smudg 




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Onion Smudjf 




Journal of Agricultural Research 



Vol. XX, No. 9 



PLATE 8 1 
Onion smudge: 

A, B, E, D. — Advanced stages of smudge after several months in storage. Note the 
shrinkage of fleshy scales and the tendency to sprout. 

C. — Bulb inoculated in a moist chamber with a suspension of Colletotrichuyn cir- 
cinans conidia. 

F, G. — Macrosporiuin sp. on outer scale of white onion sets. 

H. — M. porri and Phoma alliicola on outer scale of white onion set. Natural 
dze. 



PLATE 82 

Relation of soil temperature to the development of smudge: 

Onions kept in infected soil held at different temperature for nine days. 

A.— 5° C. 

B.— i5°C. 

C.-23° C. 

D.— 32° C. 

Slightly reduced. 



Onmn Smud? 




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Onion Smud 




Jt'urnal of Agricultural Research 



Vul^ XX, No. 9 



PLATE 83 
Colletotrichum circinans and C.fructus: 

A. — Photomicrograph of cross section of naturally infected onion scale. Note 
that the fungus is confined largely between the cuticle and the subcuticular wall. 
The epidermal cells and two layers of the parenchyma cells have collapsed, while the 
uninvaded cells beneath the lesion are slightly enlarged and distended. 

B. — Photomicrograph of cross section of an infected onion scale held for several 
months in poorly ventilated storage. Note that the stroma is excessively developed 
and that the cuticle is still intact except where ruptured by the acervuli. 

C, D. — Photomicrographs of cross sections of C. circinans (C) and C. f nidus (D) on 
apple fruit. Note similarity between the two forms and the subcuticular origin of the 
stromata in each case. 



PLATE 84 

CoUetotrichum frucius and C. circinans: 

A. — Dilution plate from spores of Colletotrick-um frucius. Photographed on sixth 
day. Note stellate character of colonies as compared with C. circinans in D. X Vs- 

B. — Individual colony of C. fructus on potato agar. Photographed on the fourth 
day. Compare with C cudmami in E. X iK^- 

C. — Apple of Fameuse variety inoculated with mycelium from pure culture of 
C. circinans. Photographed two months after inoculation. 

D. — Dilution plate from spores of C. circinans. Photographed on sixth day. Com- 
pare with C. fructus in A. X */$. 

E. — Individual colony of C. circinans on potato agar. Photographed on fourth day. 
Compare with C. fructus in B. X iK- 




Journal of Agricultural Research 



Vol. XX, No. 9 



Onion Smudge 




PLATE 85 
Relation of curing conditions to the development of smudge : 

A, B. — Comparison of onion sets artificially dried immediately after harvest with 
those not dried. Photograph made at the end of the storage period after the two lots 
had each been divided into three classes — namely, those free from disease, those 
slightly diseased, and those badly diseased. (See experiment i , p. 710-711.) 

C, D. — Comparison of white onion sets ctired in shallow crates in the field under the 
best of natural conditions with part of the same lot after exposure to moist conditions 
for one week. (See experiment 2, p. 711-712.) 



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